Cloning and overexpression of the Exiguobacterium sp. F42 gene encoding a new short chain dehydrogenase, which catalyzes the stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate

• Published in: Bioscience, biotechnology, and biochemistry Vol. 68; no. 7; pp. 1481 - 1488
• Main Authors: Wada, M. (Fukui Prefectural Univ., Matsuoka (Japan)), Yoshizumi, A, Furukawa, Y, Kawabata, H, Ueda, M, Takagi, H, Nakamori, S
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.07.2004; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Amino Acid Sequence; asymmetric reduction; Bacillaceae - enzymology; Bacillaceae - genetics; Base Sequence; Biological and medical sciences; CELL CULTURE; Chromatography, Gel; Cloning, Molecular; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; duloxetine; Electrophoresis, Polyacrylamide Gel; ESCHERICHIA COLI; Escherichia coli - genetics; Exiguobacterium; Fundamental and applied biological sciences. Psychology; GENE EXPRESSION; GENES; HORMONES; ISOLATION; MICROORGANISMS; Molecular Sequence Data; Molecular Weight; OXIDOREDUCTASES; Oxidoreductases - genetics; Oxidoreductases - metabolism; PCR; Polymerase Chain Reaction; Propionates - metabolism; RECOMBINATION; Thiophenes - metabolism; Stereoselectivity; Short chain; Reduction; Gene; Enzyme; Asymmetric reaction; Gene overexpression; duloxetine; Exiguobacterium; asymmetric reduction; Oxidoreductases; Dehydrogenase
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.68.1481

Exiguobacterium sp. F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES). (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers. The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced via an inverse PCR approach. Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family. The probable NADPH-interacting site and 3 catalytic residues (SerTyr-Lys) were fully conserved. The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E. coli by simpler procedures than from the original host. The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography. Our results show that this enzyme can be used for the practical production of (S)-HEES.